We are indebted to Xuefei Yang, Sabine Both, Lin Chen and Kaitian Wang for coordinating the fieldwork and establishing the BEF-China experiment. We are also grateful to the whole BEF-China research group for their general support. BEF-China is mainly funded by the German Research Foundation (DFG FOR 891/1 and 2) and funding for this particular project was provided by the German Research Foundation to H.B. (DFG BR 1698/9-2). We are also thankful for the travel grants and summer schools financed by the Sino-German Centre for Research Promotion in Beijing (GZ 524, 592, 698, 699 and 785). In addition we would like to thank David Eichenberg, Michael Staab, Katja Grotius, Silvana Tornack, Lin Chen, and Shouren Zhang for their support in the field and in the lab.

1.気孔コンダクタンス<ol><li>気孔コンダクタンスの測定 注：定常状態ポロメーター（十角形SC1）の単純型を利用した作者。ポロメーターのデザインは、小型、直感的な手動操作と信頼性が高いという利点があります。フィールド内の気孔コンダクタンスを測定する場合、最も効率的なことができると繰り返し測定サイクルについて測定し、個人間の距離を最小化するようにしてください。 </li><li>再現性のパターンに応じて異なる種や個人の葉（;太陽や日陰の葉など 、可能な場合のみ、同じノードから、ただ一つのカテゴリから同じ高さ、同じ露出、プラント内の同じ位置に、）を選択します。 <ol><li>唯一の対策は、健康、非損傷を受け、完全に開発された状態で残します。繰り返し測定が同じ葉の上で行われることを保証するために、（ケーブルタイや色のテープで、例えば ）個人に葉をマーク。 注：Measur気孔コンダクタンスのementsは中肋と強力な葉脈を避けながら、唯一の葉の表面になされるべきです。 </li><li>気孔コンダクタンスの値は正午に明確な減少を示すまで、5〜10を繰り返し測定を行う日の出前に早朝の時間で測定を開始します。 注：測定の毎日のコースでは、VPDと気孔コンダクタンスの関係を分析するための良好なデータをお届けします。 </li></ol></li><li> VPD測定<ol><li>好ましくは、ポータブルロガー各G Sを測定 、記録温度と相対湿度と直接同じリーフの位置の状態を測定します。蒸気圧赤字は8月、ロシュマグナス式15を使用して計算するため。 </li></ol></li></ol><img alt="式（1）" fo:content-width="3in" src="/files/ftp_upload/52738/52738eq1.jpg" width="300" /> E、S =飽和水蒸気圧[ヘクトパスカル] <ol start="4"><li>モデルのG S - VPD応答<ol><li>今種ごとに1つの分析に、個々の葉のすべての毎日のコースを組み合わせ、種ごとのVPDに対するすべてのG Sデータをプロットします。最大値を検索することにより、気孔コンダクタンスデータから観測された最大値を抽出します。 、種ごとの比較のためのモデルをスケーリングその種（G S / G SMAX）のために観察された最大値を通って観測値を分割します。 </li><li>それぞれの種について、G Sのlogitsを退行（G S / G SMAX）VPDと二項誤差分布（a、b、および回帰パラメータを表すC）と一般化線形モデルを用いて、VPDの二次項に： </li></ol></li></ol><img alt="式（2）" src="/files/ftp_upload/52738/52738eq2.jpg" width="300" /><img alt="図1" src="/files/ftp_upload/52738/52738fig1highres.jpg" wIDTH = &quot;700&quot; /&gt; 図プロットされた値とG Sに対する当てはめモデルの1例- 。VPD気孔コンダクタンスは、種フウ属のタイワン用の蒸気圧不足の関数としてプロットしました。空のドットが観察された値を表します。 （A）最大気孔コンダクタンス、最大気孔コンダクタンスでVPDと気孔コンダクタンスが絶対ので、非スケール気孔コンダクタンス（G S）データから抽出された意味。 （B）スケーリング気孔コンダクタンスデータ（G S / G SMAX）の相対パラメータ（充填ドットとして示す）20を抽出するためにプロットしました。再印刷20からの許可を得て。 <a href="https://www.jove.com/files/ftp_upload/52738/52738fig1highres.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/52738/52738fig2highres.jpg" wiDTH = &quot;400&quot; /&gt; すべての種のために、図2のすべてのフィットモデル。すべての種のVPD回帰に気孔コンダクタンスデータのモデルグラフ。エバーグリーン種は赤線20で黒線、落葉樹で表されます。 20から許可を得て再印刷します。 注：logitsを使用する代わりに、VPDに直接G Sを回帰は、グラムSMAXを超えない最大値をモデル化する点につながり、そのG Sが高いVPDで0に近づきます。 <ol start="5"><li>すべての種のための気孔規制のパラメータを抽出<ol><li>絶対モデル化されたG SMAX値 （ 図1Bの MaxFit）を計算します。これを行うには、VPD gsMaxFit = -b / 2aに与えるゼロに1.4.2の一次導関数を、設定から最大気孔コンダクタンスでVPDを計算します。 1.4.2の式にVPD gsMaxFitを挿入し、MaxFitを取得するために、電子のパワーに上げます。 Mを計算します種ごとのすべてのコンダクタンス測定のEAN（ 図1Aを参照）。 注：使用統計ソフトウェアR（http://www.r-project.org）。 </li><li>モデルの最大値（MaxFit）及び（2）VPDで（1）気孔コンダクタンスとVPD：スケールモデルからの相対値を、（G S / G SMAX）、気孔コンダクタンスとVPD値以下の2点を抽出する計算するには曲線の変曲の第二の時点で（ 図1B参照 ）。これらの点の絶対G Sの値を取得するためにグラムSMAXすることによって、これらの値を乗算します。分析した全39種の単一モデルの完全なオーバーレイについては、図2を参照してください。 </li></ol></li></ol>気孔特性の測定2。 <ol><li>気孔コンダクタンスの測定に使用されている全く同じ葉から好ましくはサンプルを取ります。これが不可能な場合、葉を選択するために適用された同一の選択手順を適用します気孔コンダクタンスを測定するため、好ましくは、同一個人に。 </li><li>新鮮なサンプルに（テスト違うが、いくつかは他よりも適しています）無色、素早く乾燥マニキュアの薄い層を適用します。サンプルは、すぐに処理できない場合は、70％アルコールに保存。マニキュアが出て乾燥した後、そっと葉からの印象をはがし、通常の葉のサンプルと同様に、顕微鏡分析のために進んでください。 注：1の酢酸溶液と酸素、過酸化物、より良い結果をもたらすことができる：水酸化ナトリウムまたは1を適用する前のステップが高いトリコーム密度の葉の場合。 </li><li>光学顕微鏡分析<ol><li> 400Xに40Xの間に倍の可能な光学顕微鏡にカメラを接続します。カメラを顕微鏡に接続された後、スケールの助けを借りて、例えば 、画像の光学倍率と解像度に撮影した画像と一致しています。 </li><li>オープンソースの私を採用ImageJの16のような魔術師処理ソフトウェアは、これらの写真を分析します。 <ol><li>いいえ汚れ、親指プリント、損傷領域、または大規模な葉脈を持つ領域の画像上の画像解析ツールからシェイプツールで形状を描画します。この領域で、サンプルあたり少なくとも50,000 平方 μmの合計で気孔をカウントします。 </li><li>気孔孔辺細胞の長さと細孔の長さを測定します。 1mm 2当たり気孔の数を計算します。 1mm 2当たりの細胞数を表皮する気孔密度の比として気孔指数を計算します。 </li></ol></li></ol></li></ol>葉脈形質の3評価注：葉脈の特性を評価するために、サック＆Scoffoni 17から修正されたプロトコルを用いました。 <ol><li>試料調製 注：葉脈の可視性を最適化するために、葉が最初に漂白した後、サフラニンとマラカイトグリーンで染色されています。 <ol><li> LEAVを漂白しますES、消色剤の50％溶液中で少なくとも72時間それらを残す（テスト違う、再びいくつかの良い仕事かもしれません）。あるいは、5％NaOH溶液、または10％のKOH水溶液または25％H 2 O 2溶液を使用します。 </li><li> 30℃にソリューションを加熱したり、より良い効果のためのステップ3.1.1からさまざまなソリューションを組み合わせます。その後水で数回洗浄します。その葉の特性に応じて、特定の種​​のための漂白工程を適応させます。 注：厚い葉は液槽およびまたはより積極的なソリューションの長い期間が必要な場合があります。シンナー、より柔らかい葉未満72時間で満足度に漂白することができます。 </li><li>葉は100％エタノールの中に置いて着色します。 1％サフラニン液に30分 - 2のためにそれらを色です。着色を強化するために、わずか数秒間1％マラカイトグリーン溶液でさらに処理を追加します。最適な結果を達成するためのタイミングと強度の面ですべての種にプロトコルを適応させます。 SEをすすぎますその後水でveral回。葉は深く染色する場合は、エタノールまたは脱色剤でいくつかの時間が役立つことがあります。 </li></ol></li><li>試料分析<ol><li> 約 1200 720dpiの解像度でバックライトスキャナと葉をスキャンします。そのピクセルの長さが戻ってスキャンした葉の絶対長さ対策にさかのぼることができる保証するために、スケールの助けを借りて、例えば 、画像の解像度に撮影したスキャンを合わせてください。 </li><li>葉の面積、外周、長さと幅を測定します。いくつかの指標、 例えば、長さ/幅と円周2 /面積を計算します。絵の真ん中から1 1 CMで長方形をカットします。 （主なミドル静脈を含まない）は、第1および第2オーダーの静脈の直径を測定します。この方形（静脈密度）の最初のオーダーのすべての静脈の長さを測定します。 </li></ol></li></ol>他の葉形質の4評価注：このような特定などアス典型的な葉の形質葉面積（SLA）、葉の乾燥物含有量（LDMC）、葉面積、要素内容、葉の習慣、葉の羽状組織、葉の化合物の種類、リーフマージンタイプなど確立したプロトコル18,19以下。 <ol><li>観測の葉の形質<ol><li>葉羽状組織、葉の化合物の種類、リーフマージンタイプとフィールドでの観察を通して花外蜜腺18,19の存在を評価します。 </li></ol></li><li>分析葉の形質<ol><li>比葉面積（SLA）の決意、葉面積と葉乾物含量（LDMC）18,19のために、好ましくは、他の測定に使用したのと同じ人に、新鮮な葉のサンプルを収集します。 80℃の乾燥オーブン中で48時間後に、好ましくは同じ葉に、要素の内容および比率を測定します。 </li></ol></li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leaf+physiology+does+not+predict+leaf+habit;+examples+from+tropical+dry+forest.">Leaf physiology does not predict leaf habit; examples from tropical dry forest.</a> Trees. 19 (3), 290-295 (2005).</li><li>Kr&#246;ber, W., Zhang, S., Ehmig, M., Bruelheide, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xylem+hydraulic+structure+and+the+Leaf+Economics+Spectrum+-+A+cross-species+study+of+39+evergreen+and+deciduous+broadleaved+subtropical+tree+species.">Xylem hydraulic structure and the Leaf Economics Spectrum - A cross-species study of 39 evergreen and deciduous broadleaved subtropical tree species.</a> PLOS ONE. 9 (11), e109211 (2014).</li><li>Kr&#246;ber, W., Heklau, H., Bruelheide, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leaf+morphology+of+40+evergreen+and+deciduous+broadleaved+subtropical+tree+species+and+relationships+to+functional+ecophysiological+traits.">Leaf morphology of 40 evergreen and deciduous broadleaved subtropical tree species and relationships to functional ecophysiological traits.</a> Plant Biology. 17 (2), 373-383 (2015).</li></ol>

The authors have nothing to disclose.

本論文で提示された方法により抽出された気孔規制のパラメータは、気孔密度と気孔指標として気孔特性の重要性を強調する。これらの新規な関係は、解剖学的および化学的な葉が20を形質、形態学的に生理的なモデルからのパラメータをリンクの可能性を示します。他の方法に比べ、本発明の方法は、気孔コンダクタンスをモデル化し、G の最大値の半分にダウンレギュレートされたユニークかつ明確なVPD値を捕捉するという利点を有します。 プロトコールに記載されているすべての手順の最も重要なものは、気孔コンダクタンスの測定値です。気孔コンダクタンスの周囲の天候条件の多因子調節にG Sに強い影響力を持っています。高い相対湿度と低照度での気孔コンダクタンスの測定は、21〜23信頼できない場合があります。形態学的およびアナトに関してomical形質は、プロトコルは常に研究に含まれる標的種に適合させる必要があります。静脈密度解析で特に、葉の漂白や染色の持続時間は、葉の構造と靭性に応じて、変化させることが必要があります。この方法の潜在的な制限は、気孔コンダクタンスの測定は不可能であるか又は複雑で異常な葉の形のためにエラーが発生しがちであるために種を含みます。これは、非常に狭い葉身と針葉樹や草を含んでもよいです。 我々の結果は、部分的に気孔コンダクタンス・パラメータおよび他のいくつかの研究に対応する葉経済スペクトル（LES）の葉の形質との間のリンクの最初の仮説を確認します。例えば、PoorterとBongers（2006）24は、G、SおよびG Sが増加葉の寿命を減少させながら、例えば LES、によって表される特性との間の密接なリンクを報告しました。したがって、シュルツら（1994）1 demonst葉の窒素含有量とg SMAXとの間に明確なリンクを評価しました。同様に、Juhrbandt ら（2004）25グラムSMAXと葉面積と葉の窒素含有率との間に有意な関係を発見しました。 葉の習慣に関して明確な違いの2つめの仮説は確認できませんでした。常緑と落葉の習慣内の測定されたパラメータと特性の高い変動は、リーフ習慣がLESの良い記述子ではありませんことを示しています。 Brodribbとホルブルック（2005）26は、広い特性変化が葉の習慣のすべてのタイプでは一般的であるため、葉の習慣と葉の生理的戦略は必然的に接続されていないことを議論しました。 アプローチは、特定の木部透水係数と顕微鏡木材は27を形質など木部の油圧に関連形質に例えば、形質および葉以外の植物器官の生理学的特徴に拡張することができます。同様に、他のこのような柵状柔構造とエピクチクラワックス層構造として顕微鏡由来として葉の形質の種類は28を含めることができます。 要約すると、本研究では、LESと気孔調節との間の密接な関係を確認しました。また、ここで紹介する方法は、LESに関連していない気孔規制パターンの側面を明らかにしました。このような気孔の大きさ、密度、インデックスだけでなく、静脈長として特に特定の葉の形質には機能的な植物の研究に今後注目に値します。

植物の葉の機能の理解を進めるために、多くの最近の研究は、葉の気孔コンダクタンス（GのS）1-4のような生理的反応、形態学的に、解剖学的および化学葉の形質に関連することを試みてきました。また、葉の形質に、気孔コンダクタンスが強く、このような光合成有効光量子束密度、空気の温度とVPD 5のような環境条件によって影響されます。主にVPD 6のGSの線形回帰に基づいているVPD曲線6-8 -様々な方法がGSをモデル化するために提案されています。これとは対照的に、本 ​​研究で提示したモデルは、VPDに（最大気孔コンダクタンスG SMAXにG Sのすなわち、比率）の相対的な気孔コンダクタンスのlogitsを退行し、二次回帰の用語としてVPDを追加することにより、非直線性を占めています。 他のモデルと比較して、新しいモデルが記述するパラメータを導出することができますG Sは水不足の下でダウンレギュレートされたVPD。同様に、VPDがG Sが最大となるで得られます。そのような生理学的パラメータは、LESに反映されるようにしっかりと炭素同化9,10栄養や資源配分にこれらのモデルパラメータとキー葉形質との間の密接なリンクにリンクすることが期待できるとして3,11期待されるべきです。その結果、またLES形質を持つ気孔規制の戦略との間に密接な関係があるはずです。このような関係は、双方のLESおよび水利用効率12,13と相関している葉の習慣として（常緑対落葉）の葉の習慣のために特に期待されています。エバーグリーン種は遅く成長する傾向があるが、栄養分14の貧しい環境でより効率的です。このように、葉の習慣が落葉の種よりも保守的な水利用戦略で、気孔規制パターンが異なるに変換する必要があります。 コンプ共通の庭の状況で広葉樹の種の大規模なセットをARING、次の仮説を試験した：G Sからの1）モデルのパラメータは- VPDモデルは、葉の経済スペクトルに関連した葉の形質に接続されています。 2）エバーグリーン種は落葉種よりも低い平均G SおよびG SMAXの値を持ちます。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>SC 1 Porometer</td>
			<td>Decagon</td>
			<td>NA</td>
			<td>Any other porometer is suitable</td>
		</tr>
		<tr>
			<td>Cable ties to mark leaves</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Plastic sample bags</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Paper sample bags</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Hygrometer</td>
			<td>Trotec</td>
			<td>NA</td>
			<td>Any other is suitable</td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Axioskop 2 plus</td>
			<td>Zeiss</td>
			<td>NA</td>
			<td>Any other is suitable</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>5% NaOH</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>10% KOH</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>25% H2O2</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Malachite green</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Safranine</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
	</tbody>
</table>

relating stomatal conductance to leaf functional traits

彼らはそのような蒸散と炭素同化などの生理学的機能を反映するため、葉の機能特性が重要です。具体的には、形態学的形質の葉は、成長パターン及び栄養利用、水利用効率の観点から、植物戦略を要約する可能性があります。葉の経済スペクトル（LES）は、機能植物生態学における認識のフレームワークであり、（特定の葉面積（SLA）、葉の窒素、リン、陽イオン含有量を増加し、葉の乾物含量（LDMC）と炭素窒素比を低下させる勾配を反映していますCN）。 LESは、葉の質量あたりの光合成能力と短命の葉のことから、低質量系炭素同化率と長寿命の葉に至るまで様々な戦略について説明します。しかし、LESに含まれていない形質は、このような気孔制御に関連するものとして、種の生理機能に関する追加情報を提供することがあります。プロトコルは、葉の広い範囲のために提示されていfunctioLESの特性だけでなく、LESとは独立した形質を含む最終的形質。具体的には、新しい方法は、蒸気圧の赤字に気孔コンダクタンスの工場 &#39;調節行動に関するものが導入されます。気孔調節の結果として得られるパラメータは、LES及び他の植物の機能的特性と比較することができます。結果は、LESの機能葉の形質は気孔規制のパラメータにも有効な予測因子であったことを示しています。例えば、葉の炭素濃度は、屈曲及びコンダクタンスVPD曲線の最大の点で蒸気圧不足（VPD）に積極的に関連していました。しかし、LESに含まれていない形質は気孔制御のパラメータを説明する際に情報を追加しました：コンダクタンス-VPD曲線の変曲点でのVPDは、より高い気孔密度と高い気孔指数を有する種のために低かったです。全体的に、気孔および静脈形質は気孔規制トンを説明するためのより強力な予測因子でしたLESで使用漢特性。

気孔コンダクタンスと気孔調節の多くのパラメータは、形態学的解剖学的および化学的な葉形質に関連することが見出されました。以下では、焦点は気孔密度（P = 0.04）と気孔率（P = 0.03）で減少し、 図を参照してください 、葉の炭素含有量（P = 0.02に増加した変曲点、でVPDへのリンクになります3）。結果は、VPDの減少に伴って変曲点で気孔密度と気孔指数の低下があったことを示しています。これとは対照的に、気孔コンダクタンスのないパラメータは、葉の習慣に明確な関係は認められませんでした。葉の習慣の二つのグループ内の高変動は異なる調節機構は常緑と落葉の習慣のグループ内の両方を存在することを示しています。 <img alt="図3" src="/files/ftp_upload/52738/52738fig3highres.jpg" width="700" /> 番目の図3.結果図気孔コンダクタンスの規制や葉形質のパターン間の電子リンク蒸気圧の赤字（VPD）G Sの変曲点で- 。（A）気孔密度、（B）気孔指数の関数としてVPD曲線（VpdPoi）、および（C）葉の炭素含有量20。再印刷20からの許可を得て。 <a href="https://www.jove.com/files/ftp_upload/52738/52738fig3highres.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Relating Stomatal Conductance to Leaf Functional Traits at JoVE.com

Relating Stomatal Conductance to Leaf Functional Traits

生理学と形態がリンクされている方法を解明することは、植物の葉のメカニズムの機能をより深く理解することができます。私たちは、伝統的な機能の葉形質と気孔コンダクタンスの測定値との相関関係から気孔規制のパラメータを導出するための手順の両方を提示します。

葉の機能上の特徴に気孔コンダクタンスの関連付け

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, ...

relating-stomatal-conductance-to-leaf-functional-traits

Research

JoVE Journal

Biology

19.0K ビュー.  Martin-Luther-University Halle-Wittenberg. 生理学と形態がリンクされている方法を解明することは、植物の葉のメカニズムの機能をより深く理解することができます。私たちは、伝統的な機能の葉形質と気孔コンダクタンスの測定値との相関関係から気孔規制のパラメータを導出するための手順の両方を提示します。 

ビデオ: Relating Stomatal Conductance to Leaf Functional Traits

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

マウス用ダーク/ライト遷移テスト

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

生物学

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

脂質中間相を用いた構造決定のための膜タンパク質の結晶化

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

胚形成の間に機能的な冗長性をテストするための逆遺伝学的アプローチ

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

で嚢胞性線維症膜コンダクタンス制御因子タンパク質の発現および精製サッカロマイセスセレビシエ

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method (EFM)

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to achieve a high stomatal conductance (gs) to capture CO2 for photosynthesis, water is lost by transpiration1,2. Water evaporating from the airspaces is replaced from cell walls, in turn drawing water from the xylem of leaf veins, in turn drawing from xylem in the stems and roots. As water is pulled through the system, it experiences hydraulic resistance, creating tension throughout the system and a low leaf water potential (&Psi;leaf). The leaf itself is a critical bottleneck in the whole plant system, accounting for on average 30% of the plant hydraulic resistance3. Leaf hydraulic conductance (Kleaf = 1/ leaf hydraulic resistance) is the ratio of the water flow rate to the water potential gradient across the leaf, and summarizes the behavior of a complex system: water moves through the petiole and through several orders of veins, exits into the bundle sheath and passes through or around mesophyll cells before evaporating into the airspace and being transpired from the stomata. Kleaf is of strong interest as an important physiological trait to compare species, quantifying the effectiveness of the leaf structure and physiology for water transport, and a key variable to investigate for its relationship to variation in structure (e.g., in leaf venation architecture) and its impacts on photosynthetic gas exchange. Further, Kleaf responds strongly to the internal and external leaf environment3. Kleaf can increase dramatically with irradiance apparently due to changes in the expression and activation of aquaporins, the proteins involved in water transport through membranes4, and Kleaf declines strongly during drought, due to cavitation and/or collapse of xylem conduits, and/or loss of permeability in the extra-xylem tissues due to mesophyll and bundle sheath cell shrinkage or aquaporin deactivation5-10. Because Kleaf can constrain gs and photosynthetic rate across species in well watered conditions and during drought, and thus limit whole-plant performance they may possibly determine species distributions especially as droughts increase in frequency and severity11-14.
We present a simple method for simultaneous determination of Kleaf and gs on excised leaves. A transpiring leaf is connected by its petiole to tubing running to a water source on a balance. The loss of water from the balance is recorded to calculate the flow rate through the leaf. When steady state transpiration (E, mmol &bull; m-2 &bull; s-1) is reached, gs is determined by dividing by vapor pressure deficit, and Kleaf by dividing by the water potential driving force determined using a pressure chamber (Kleaf= E /- &Delta;&psi;leaf, MPa)15.
This method can be used to assess Kleaf responses to different irradiances and the vulnerability of Kleaf to dehydration14,16,17.

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method EFM

We describe a relatively rapid (30 min) and realistic method for simultaneously measurement of leaf hydraulic conductance (Kleaf) and stomatal conductance (gs) for transpiring excised leaves. The method can be modified to measure the light and dehydration responses of Kleaf and gs.

蒸発フラックス法（EFM）を使用した照度と脱水に葉の通水コンダクタンスと気孔コンダクタンスとその応答の測定

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to ...

LeafJ: An ImageJ Plugin for Semi-automated Leaf Shape Measurement

High throughput phenotyping (phenomics) is a powerful tool for linking genes to their functions (see review1 and recent examples2-4). Leaves are the primary photosynthetic organ, and their size and shape vary developmentally and environmentally within a plant. For these reasons studies on leaf morphology require measurement of multiple parameters from numerous leaves, which is best done by semi-automated phenomics tools5,6. Canopy shade is an important environmental cue that affects plant architecture and life history; the suite of responses is collectively called the shade avoidance syndrome (SAS)7. Among SAS responses, shade induced leaf petiole elongation and changes in blade area are particularly useful as indices8. To date, leaf shape programs (e.g. SHAPE9, LAMINA10, LeafAnalyzer11, LEAFPROCESSOR12) can measure leaf outlines and categorize leaf shapes, but can not output petiole length. Lack of large-scale measurement systems of leaf petioles has inhibited phenomics approaches to SAS research. In this paper, we describe a newly developed ImageJ plugin, called LeafJ, which can rapidly measure petiole length and leaf blade parameters of the model plant Arabidopsis thaliana. For the occasional leaf that required manual correction of the petiole/leaf blade boundary we used a touch-screen tablet. Further, leaf cell shape and leaf cell numbers are important determinants of leaf size13. Separate from LeafJ we also present a protocol for using a touch-screen tablet for measuring cell shape, area, and size. Our leaf trait measurement system is not limited to shade-avoidance research and will accelerate leaf phenotyping of many mutants and screening plants by leaf phenotyping.

Demonstration of key methods for high throughput leaf measurements. These methods can be used to accelerate leaf phenotyping when studying many plant mutants or otherwise screening plants by leaf phenotype.

LeafJ：半自動化された葉の形状計測のためのImageJのプラグイン

High throughput phenotyping (phenomics) is a powerful tool for linking genes to their functions (see review1 and recent examples2-4). Leaves are the ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

で表現する嚢胞性線維症膜コンダクタンス制御因子タンパク質の精製<em&gt;サッカロマイセス·セレビシエ</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

タンパク質 - タンパク質相互作用を検出するために、高密度機能タンパク質マイクロアレイのプロービング

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular systolic and diastolic function. Measures of ventricular contractility and compliance are obtained through examination of cardiac response to changes in afterload and preload. These techniques were originally developed nearly three decades ago to measure cardiac function in large mammals and humans. The application of these analyses to small mammals, such as mice, has been accomplished through the optimization of microsurgical techniques and creation of conductance catheters. Conductance catheters allow for estimation of the blood pool by exploiting the relationship between electrical conductance and volume. When properly performed, these techniques allow for testing of cardiac function in genetic mutant mouse models or in drug treatment studies. The accuracy and precision of these studies are dependent on careful attention to the calibration of instruments, systematic conduct of hemodynamic measurements and data analyses. We will review the methods of conducting pressure-volume loop experiments using a conductance catheter in mice.

Cardiac pressure-volume loop analysis is the most comprehensive way to measure cardiac function in the intact heart. We describe a technique to perform and analyze cardiac pressure volume loops, using conductance catheters.

マウスにおけるコンダクタンスカテーテルを用いて心臓圧力 - 体積ループ解析

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular ...

Fish Sperm Assessment Using Software and Cooling Devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

魚の精子の評価ソフトウェアを使用して、冷却装置

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...